1. Introduction

Biofilm formation commonly associated with many infections and health problems are usually a serious concern for clinicians because of their resistance to a wide range of antibiotics ^{[1, 2]} and to clearance by humoral or cellular host defense mechanisms ^[3]. It has been estimated that 65-80% of all human infections are biofilm-related ^[4]. The problem of treating biofilm-associated infections is not only attributed to the difficulty of eradicating the biofilm focus but also to the lack of susceptibility of cells disrupted from the biofilm to antimicrobial agents ^[2]. Because of the evident relationship between various infectious diseases and the presence of the microorganisms in the biofilm, there has been an escalation in the interest in studying the microbial biofilm and its impact in human health.

In Situ and in vivo experimental studies of the biofilm in humans are difficult to approach due to limitation in accessing and sampling, the difficulties of standardization and control of assay conditions as well as the complicated ethical aspects ^[5]. Animal models, on the other hand, are useful in studying the pathogenicity and to search for compounds with therapeutic potential against biofilm ^{[6, 7, 8]}.

Starting with the simple microtiter plate assay and scanning electron microscopy, tools for studying biofilm formation in vitro have since evolved to include specialized equipments such as flow cell systems and real-time analysis using reporter gene assays ^[9]. Several in vitro models have been described to study the formation of microbial biofilms and the factors that affect their behavior under static or dynamic conditions. The tube test as well as microtiter plate assay were among the earliest methods described for assessing microbial biofilm formation under static system ^{[10, 11]}. Variations in data between and within different laboratories when using the tube test and the microtiter plate assay have raised a lot of concerns ^[11]. In addition, a number of ELISAs (Enzyme-linked immunosorbent assays) for investigating factors influential in biofilm have been also developed ^[12].

Although they are routinely used when evaluating biofilm structure and susceptibility to antimicrobial agents, in vitro models that adopt the static system seem to be misleading if compared to the real dynamic condition. Static system does not take into account key factors such as the fluid shear stress which affects the adhesion and biofilm formation by microorganisms.

One of the most used dynamic models to study the biofilm mode of growth is the flow cell system ^[7]. This system was first developed as Robbins Device (RD), modified Robbins Device (MRD) ^[13] and recently as commercially available flow cells ^{[14, 15, 16]}. Most of the commercially available devices are expensive, have minimum configuration limits and are designed to test the biofilm on small segments of the catheter materials rather than to use the whole catheter. To simulate the real environment of the biofilm on catheters, it would be advantageous for the design of the model to allow for the insertion of the catheter in its original state. The physical configuration of the studied catheter such as the diameter and length are important especially when studying the effect of fluid flow on biofilm.

The aim of this work is to develop a simple in vitro novel device which mimics the real life of the biofilm formation and could be modulated to contain most catheter and tubes and readily allows biofilm formation under different experimental conditions.

2. Materials and Methods

Unless otherwise indicated, all chemicals (analytical grade) were purchased from Sigma-Aldrich, USA.

Two clinical isolates, Staphylococcus epidermidis and Candida albicans, were used to validate the ability of the device to support biofilm formation on intravenous (IV) catheter. The microorganisms were isolated from bloods of patients with central venous catheters. S. epidermidis was identified using conventional microbiological techniques and C. albicans was identified to species level by using API 20 C AUX for yeast (bioMérieux).

The device comprises a tubular body defining a test chamber (Figure 1 & supplemented video file). The size of the test chamber is of varied height within the range of about 10-50 cm, and the diameter is in the range of about 0.8-2.5 cm. The volume of the test chamber is in the range of about 5-20 ml. The body has upper and lower ends provided with closures. Each of the end has one port which can be used as an outlet or inlet based on the studied conditions. The body has a built in side port with the all three ports can be connected to a tubing system or blocked by the removable closures. The ports in the upper and lower ends of the device are designed to mount the tested catheter. The design allows the fluid to be pumped through the inner lumen of the implant tube before filling the inner chamber to allow biofilm formation on the inner and outer surfaces of the catheters.

Figure 1. Design of the biofilm device

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The various parts of the device may be fabricated from stainless steel, Pyrex glass or other materials which do not affect the formation of the biofilm.

The device can be set up flexibly in different configurations to suit the studied conditions as follow:

The device can be configured to test the biofilm under static condition by simply using it as a closed system (Figure 2A). Ports 2 and 3 are blocked while port 1 is used to inoculate the chamber and provide the system with the culture medium.

The biofilm can be kept under continuous flow of fresh medium provided from a reservoir via IV infusion pump and IV tubing set. The medium is allowed to flow through the lumen of the catheter before passing to the chamber. Port 1 is used as the inlet, port 2 is blocked while port 3 is used to collect the overflowed liquid which contain planktonic and cells dispersed from the biofilm (Figure 2B).

The device and reservoir were sterilized by autoclaving before insertion of the catheter and connection to the infusion pump via sterile IV tubing set. During experiment, the whole system was kept in laminar flow cabinet to avoid contamination.

Figure 2. Configuration of the biofilm device under static (A )and dynamic fluid flow systems (B)

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The device was configured to test biofilm formation of S. epidermidis and C. albicans under static and dynamic conditions as mentioned above. For biofilm formation under static condition, 24 hours old culture of S. epidermidis on Tryptic Soya Agar (TSA) or C. albicans on Sabouraud Dextrose (SAB) agar medium were used to inoculate Tryptic Soya Broth (TSB) or Yeast Nitrogen Base (YNB) media supplemented with 2% dextrose respectively. The initial inoculum size was standardized in the media to give 1-5 X 10⁶ CFU/ml of the tested microorganisms. Peripheral venous catheter BD Angiocath, reference number 382259 (Becton, Dickinson and Company, USA) was placed in the sample insert. The culture medium, with either microorganism, was pumped into the catheter to fill the chamber. The device was then incubated at 37^oC for 48 hours after which the catheter was washed by passing sterile phosphate buffered saline (PBS) (pH 7.3) at 30 ml/hour for 2 hours to remove the planktonic (free-floating) cells. The catheter was aseptically removed using sterile forceps and cut to 1 cm segments in microbiological safety cabinet. The biofilms on the catheters were washed and dislodged by sonication in 1 ml ice-cooled PBS at 30% cycle, 3.5 output for 30 seconds followed by vortexing for another 30 seconds. The number of sessile cells was determined by viable counts on SAB agar or TSA medium and calculated as colony forming unit (CFU)/cm²of the catheter surface.

In another set of experiments, the device was configured to test the biofilm formation under dynamic condition. Briefly, the culture media, supplemented with 1-5 X 10⁶ CFU/ ml of the tested isolate, was delivered to the catheter at 30 ml/h for 2 h. The planktonic cells were removed by delivering sterile media at the same rate for another 2 h. The biofilms on the catheter were then kept under continuous flow of fresh medium at 10 ml/ h for 48 hours. The catheters were then cut to 1 cm segments, and the biofilms were dislodged and sessile cells were determined as previously mentioned.

The device was compared to biofilm devices which support biofilm formation under static condition, the disc model ^[17], and continuous fluid flow system, the MRD.

The Scanning Electron Microscope (SEM) was used to visualize the biofilm architecture of S. epidermidis and C. albicans which formed on the vascular catheters under dynamic conditions. Catheters, from the experiment in which the biofilms were formed under continuous flow of the medium, were aseptically cut to 1cm segments and prepared for Scanning Electron Microscope examination as previously described ^[18]. Briefly, they were fixed in glutaraldehyde in 0.1 M cacodylate buffer containing 0.15 ruthenium red for 3 h at 4°C. The segments were then rinsed in fresh 0.1 M cacodylate buffer for 10 min (repeated three times) and post-fixed in 1.5 % osmium tetroxide for 1 h. They were dehydrated in a series of aqueous ethanol solutions (30–100 %) and dried by a critical point dryer (Autosamdri) with CO₂. The specimens were mounted on aluminium stubs with silver paste, allowed to dry for 3 h and then coated with gold/palladium using a cool-sputter coater E5100 II (Polaron Instruments). The segments were then examined in a SEM (S-500; Hitachi) at 20 kV.

The viability of S. epidermidis cells within the biofilm environment was demonstrated using Confocal Scanning Laser Microscopy (CSLM). Catheter segments on which the biofilms were formed under continuous flow of the medium were used. Briefly, the catheter segments were transferred to a tube containing 1 ml ice-cooled saline and the adherent cells were dislodged by sonication. Hundred microliter portion of the suspension was transferred to a cover slip, and the dislodged cells were stained with LIVE/DEAD BacLight bacterial viability stain (Molecular Probes, Eugene, OR, USA) following manufacturer's instructions. Finally, the sample was examined by Olympus Fluoview CSLM (model IX 70, Olympus America Inc. NY, USA).

The shear stress on the inner and outer surfaces of the catheter was determined at different flow rates of the culture medium (1-100 ml/h). The flow type is either laminar or turbulent based on the value called Reynold’s number (R_e), where R_e is calculated using the following equation: ^[19].

Where ρ and µ are the fluid density and viscosity, u is the velocity and D is the catheter diameter.

The shear stress can be calculated using the following equation if the flow is laminar:

Where k is a constant which depends on the catheter diameter, fluid density and viscosity and Q is the volume flow rate (milliliter/hour).

Each experiment of the biofilm formation was performed in quadruplicate and the mean and the Standard Deviation (S.D.) were calculated. One-way analysis of variance (ANOVA) was used to determine the differences between various treatments. Tukey’s pair comparison test was used at the chosen level of probability (P<0.05) to determine significance difference between means.

3. Results

The biofilms of the tested microorganisms on the catheter were evaluated quantitatively by viable count technique and semi-quantitively by visualizing the biofilm communities by SEM and CSLM. The device was found to support the biofilm formation of the tested bacteria and yeast under both static and continuous fluid flow systems (Figure 3).

Figure 3. Validation of the device to support biofilm formation

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When comparing the means of log values of microorganisms contained in the biofilms, difference in the number of adherent cells was demonstrated in the two systems. For S. epidermidis, the log value of the number of cells contained in the biofilm under static system was 6.41 ± 0.22. This value was significantly higher (p <0.001) than that of the biofilm which formed under dynamic system (5.18 ±0.13). Significant population variability (p = 0.012) was also observed with the biofilm of C. albicans where the log value of the number of cells was 6.44 ± 38 and 5.47 ± 0.05 under static and dynamic conditions respectively.

The device was compared to two biofilm models devices that adopt static or continuous fluid flow system under the same tested experimental condition, the disc and MRD respectively. The device showed better support to the biofilm at the tested conditions (Table 1).

Table 1. Comparison of the device to other biofilm-supporting devices

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Further endorsing evident for the ability of the device to support biofilm formation was also concluded from SEM (Figure 4 A & B). The biofilm of S. epidermidis appeared as homogenous thick layers while the biofilm of C. albicans consisted predominantly from filamentous growth with few yeast cells. Similarly, CLSM (Figure 5) demonstrated the viability of the majority of S. epidermidis cells, stained with green fluorescence, within the disrupted biofilm compared to low number of dead cells stained with red florescence.

Figure 4. SEM showing the biofilm architecture of Streptococcus epidermidis (A) and C. albicans (B) on vascular catheter segment using the biofilm device

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Figure 5. Visualization of the viability of S. epidermidis cells within the biofilm environment using Confocal Scanning Laser Microscopy (CSLM)

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Figure 6. Determination of the fluid shear stress on the inner and outer surface of catheter at different flow rate of the media

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In order to measure the shear stress on the biofilm, Reynold’s number (R_e) was determined at different flow rates of the culture medium. At 1-100 ml/h, the recorded R_e values were in the range of 0.35 - 35.24 and 0.035- 3.52 on the inner and outer surfaces of the catheter respectively, which indicates a low laminar flow system. Based on the laminar flow system, the shear stress was determined on the inner and outer surfaces of the catheters where the values were in the range of 0.0028- 0.28 and 4.01 x 10^-5- 0.004 N/ m² respectively (Figure 6 A & B).

4. Discussion

Biofilm model systems are essential to understand the development, behavior and the nature of the microbial communities within the biofilm and the mechanism of their resistance to antimicrobial agents and host defense system. In vitro biofilm models are important tools in experimental medical science to better understand biofilm physiology and micro-ecology ^[5]. Such models may be used to simulate environmental conditions within the laboratory or to focus on selected variables such as growth rate or fluid flow ^[20].

The presented biofilm device is designed to mimic the real environment of the biofilm. Its design makes it easy to contain a full length catheter in the main chamber instead of using segments or piece of plastics as in other available devices. The device can also be adjusted to different configurations to fit the tested experimental conditions required to simulate and study microbial biofilm by controlling the fluid flow through the different ports. The two ends of the device are detachable and come with different sizes of sample insert to fit most types of catheters.

The use of venous catheters is well known as the leading cause responsible for health care–associated infections ^{[21, 22, 23]}. Therefore, IV Catheters were used in this study to test the ability of the device to support microbial adherence and biofilm formation. Catheter-related bloodstream infections affect more than 200,000 patients per year in the United States ^{[24, 25]}. These infections are associated with an increased risk of death, with increases in morbidity, length of stay, and health care costs ^[22].

Two microorganisms, S. epidermidis and C. albicans, were selected to validate the device. Among the cogulase-negative staphylococci (CNS), S. epidermidis is the most frequently isolated species and the most common species responsible for infection ^[26]. Infections caused by S. epidermidis are often persistent and relapsing. S. epidermidis and other CNS are generally the causative organisms in the majority of device-related infections ^[27]. C. albicans, on the other hand, is the fourth leading cause of vascular catheter-related infections and the third leading cause of urinary catheter-related infections ^{[28, 29]}. Among vascular catheter-related infections, those due to Candida spp. are associated with the highest rate of mortality ^[30].

The device was found to support the biofilm formation of the tested bacteria and yeast under both static and continuous fluid flow systems (Figure 3). The results were comparable to what have been published before using other in vitro biofilm devices ^{[2, 31]}. When compared to the disc model and the MRD, the device showed better support to the biofilm at the tested conditions (Table 1). The number of cells contained in the biofilm under static system was significantly higher than that of the biofilm which formed under dynamic system for both microorganisms. The difference could be attributed to the nature of the static system where factors such as longer contact time and sedimentation of the microorganism by gravity force could allow the microorganisms to better adhere and colonize the catheter surface. On the contrary, in continuous fluid flow system, factors such as fluid flow rate and wall shear stress may affect the adhesion, and detachment of microbial cells ^[5].

The ability of the device to support the biofilm formation was demonstrated using SEM (Figure 4 A & B) and CLSM (Figure 5). The micrographs showed the biofilm of S. epidermidis as homogenous thick layers while the biofilm of C. albicans as predominant filamentous growth with few yeast cells. C. albicans has both a budding and filamentous forms. Both forms are important for invasion and biofilm formation. The yeast form is important for dissemination in blood whereas the filamentous form is suited for invasion and propagation on tissues and surfaces. C. albicans biofilms incorporating both yeast and filamentous forms, with budding yeast attached to the surface and the filamentous form on top. The CLSM, on the other hand, demonstrated the viability of the majority of S. epidermidis cells within the disrupted biofilm compared to low number of dead cells.

Based on recorded Reynold’s number (R_e), the design of the presented device permits low laminar flow system with very low shear stress on the inner and outer catheter surfaces (Figure 6 A & B). The wall share stress inside the flow cells is directly influencing the process of biofilm formation. It depends on the fluid flow rate (ml/h), fluid velocity (mm/s), and dimensions of the system, and acts as the main source of fluid flow forces on adhering organisms ^[32]. The shear stress on the inner and outer surfaces of the catheter was determined at different flow rates of the culture medium (1-100 ml/h). Various flow rate values have been mentioned in the literatures with regard to the different scientific applications of flow cell systems to simulate biofilm formation in different sites of the human body ^{[33, 34, 35]}. Liquid flow can be described as either laminar or turbulent based on the Reynold’s number (R_e), a function of the density, viscosity, linear distance traveled and velocity of a flowing fluid. R_e can be used to describe the hydrodynamics of a flowing system. In flowing systems with a low Re, laminar flow may exist with little or no mixing. In turbulent flow, the liquid in the plug is rapidly mixed. Laminar flow is usually characterized by low fluid velocity (Re < 2100) while high fluid velocity generally leads to turbulent flow (R_e > 4000) ^[19].

Increased fluid flow towards or parallel to a substratum surface results in faster adhesion of microorganisms due to higher mass transport ^[36]. A higher fluid shear, on the other hand, may lead to their detachment ^[37] and a very high value would prevent the adherence of microbial cells and biofilm formation ^[38].

5. Conclusions

The presented device is simple and can be fabricated from cheap materials. It is not only simulating the real biofilm environments, but also could be modulated to contain most catheter and tubes and readily allows biofilm formation under different experimental conditions. The design permits low laminar flow system which is common in studying biofilms in flow cell systems. Accordingly, it is well suited to study the real life of the biofilm formation by bacteria and yeasts.

Acknowledgment

- The author would like to thank the following colleagues for their help and contribution:

-Dr. Nancy Khardori, Professor of Internal Medicine, School of Medicine, Southern Illinois University, USA, for her guidance during designing of the device.

-Dr. Hamdy A. Kandil, Associate Professor, Mechatronics Engineering Department, German University in Cairo, for his help in calculating the R_e values and shear stress on the tubing system within the device.

- Dr. Mohamed Harraz, Lecturer at the Department of Engineering Design and Production Technology, Faculty of Engineering and Material Science, and Engineer Ahmed Wagdy, Teaching and Research Assistant at the Faculty of Engineering and Material Sciences for their help in generating the 2-D figure and 3-D animation of the device.

List of Abbreviations

ELISAs: Enzyme-linked immunosorbent assays

RD: Robbins Device

MRD: modified Robbins Device

IV: Intravenous

TSA: Tryptic Soya Agar

SAB: Sabouraud Dextrose

TSB: Tryptic Soya Broth

YNB: Yeast Nitrogen Base

CFU: Colony forming unit

SEM: Scanning Electron Microscope

CSLM: Confocal Scanning Laser Microscope

CNS: Cogulase-negative staphylococci.

Competing Interest

The authors declare that he has no competing interests.

References

[1]	Prosser, P.L., Taylor, D., Dix B.A. and Cleeland, R, “Method of evaluating effects of antibiotics on bacterial biofilm,” Antimicrob Agents Chemother, 31(10). 1502-1506. Oct.1987.
	In article
[2]	El-Azizi, M., Rao, S., Kanchanapoom, T. and Khardori N, “In vitro activity of vancomycin, quinupristin/dalfopristin, and linezolid against intact and disrupted biofilms of staphylococci,” Ann Clin Microbiol Antimicrob, 4(2). Jan. 2005 [Online].
	In article
[3]	Johnson, G.M., Lee, D.A., Regelmann, W.E., Gray, E.D., Peters, G. and Quie, P.G, “Interference with granulocyte function by Staphylococcus epidermidis slime,” Infect Immun, 54(1). 13-20. Oct.1986.
	In article
[4]	Coenye, T. and Nelis, H.J, “In vitro and in vivo model systems to study microbial biofilm formation,” J Microbiol Methods, 83(2). 89-105. Nov.2010.
	In article
[5]	Pavarina, A.C., Dovigo, L.N., Sanita, P.V., Machada, A. L., Giampaolo, E.T. and Vergani, C.E, Biofilm formation: development and properties, Nova Science Publishers Inc., New York, 2011, 125-162.
	In article
[6]	Andes, D., Nett, J., Oschel, P., Albrecht, R., Marchillo, K. and Pitula, A, “Development and characterization of an in vivo central venous catheter Candida albicans biofilm model,” Infect Immun, 72(10). 6023-6031.Oct.2004
	In article
[7]	Freire, M.O., Sedghizadeh, P.P., Schaudinn, C., Gorur, A., Downey, J.S., Choi, J.H., Chen, W., Kook, J.K., Chen, C.,Goodman, S.D. and Zadeh, H.H, “Development of an animal model for Aggregatibacter Actinomycetemcomitans biofilm-mediated oral osteolytic infection: A preliminary study,” J Periodontol, 82(5). 778-789. May 2011.
	In article
[8]	Williams, D.L. and Costerton, J.W, “Using biofilms as initial inocula in animal models of biofilm-related infections,” J Biomed Mater Re, 100(4). 1163-1169.May 2012.
	In article
[9]	Harraghyi, N., Seiler, S., Jacobs, K., Hannig, M., Menger, M.D. and Herrmann, M, “Advances in in vitro and in vivo models for studying the staphylococcal factors involved in implant infections,” The International Journal of Artificial Organs, 29(4). 368-378. Apr. 2006.
	In article
[10]	Christensen, G.D., Simpson, W.A., Younger, J.J., Baddour, L.M., Barrett, F.F. and Melton, D.M, “Adherence of coagulase negative staphylococci to plastic tissue culture plates: A quantitative model for the adherence of staphylococci to medical devices,” J Clin Microbiol, 22(6). 996-1006. Dec. 1985.
	In article
[11]	Stepanovic, S., Vukovic, D., Dakic, I., Savic, B. and Svabic-Vlahovic, M, “A modified microtiter-plate test for quantification of staphylococcal biofilm formation,” J Microbiol Methods, 40(2). 175-179. Apr. 2000.
	In article
[12]	Mack, D., Siemssen, N. and Laufs, R, “Parallel induction by glucose of adherence and a polysaccharide antigen specific for plastic-adherent Staphylococcus epidermidis: Evidence for functional relation to intercellular adhesion,” Infect. Immunol, 60(5). 2048-2057. May 1992.
	In article
[13]	McCoy, W.F., Bryers, J.D., Robbins, J. and Costerton, J.W, “Observations in fouling biofilm formation,” Can J Microbiol, 27 (9).910-917. Sep. 1981.
	In article
[14]	Leung K.P., Crowel, T.D., Abercrombie, I.J., Molina, C.M., Bradshaw, C.J., Jensen, C.L., Luo, Q. and Thompson, G.A, “Control of oral biofilm formation by an antimicrobial decapeptide,” J Dent Res, 2005, 84(12). 1172-77. Dec. 2005.
	In article
[15]	Chin, M.Y., Busscher, H.J., Evans, R., Noar, J. and Pratten, J, “Early biofilm formation and the effects of antimicrobial agent in orthodontic bonding materials in a parallel plate flow chamber,” Eur J Orthod, 28(1). 1-7. Feb.2006.
	In article
[16]	Rieu, A., Briandet, R., Habiana, O., Garmyn, D., Guzzo, J. and Piveteau, P, “Listeria monocytogenes EGD-e biofilms: no mushroom but a network of knitted chains,” Appl Environ Microbiol, 74(14). 4491-4497. Jul. 2008.
	In article
[17]	El-Azizi, M, “Enhancement of the in vitro activity of amphotericin B against the biofilms of non-albicans Candida spp. by rifampicin and doxycycline,” J Med Microbiol, 56(Pt5). 645-649. May 2007.
	In article
[18]	Marrie, J. and Casterton, W, “Scanning and transmission electron microscopy of in situ bacterial colonization of intravenous and intraatrial catheters,” J Clin Microbiol, 19(5). 687-693. May 1984.
	In article
[19]	Cengel, Y.A. and Cimbala, J.M, Fluid Mechanics: Fundamentals and Applications. 1st edition, McGraw Hill, New York, 2006, 322-325.
	In article
[20]	McBain, A.J, “In vitro biofilm models: An overview,” Adv Appl Microbiol, 69. 99-132. 2009.
	In article
[21]	Wenzel, R.P. and Edmond, M.B, “The impact of hospital-acquired bloodstream infections,” Emerg Infect Dis, 7(2). 174-177. March-Apr. 2001.
	In article
[22]	Eggimann, P., Sax, H. and Pittet, D, “Catheter-related infections,” Microbes Infect, 6(11).1033-1042. Sep. 2004.
	In article
[23]	Pittet, D, “Infection control and quality health care in the new millennium,” Am J Infect Control, 33(5). 258-267. Jun 2005.
	In article
[24]	Mermel, L.A, “Prevention of intravascular catheter-related infections,” Ann Intern Med, 132(5). 391-402. March 2000.
	In article
[25]	Darouiche, R.O, “Device-associated infections: a macroproblem that starts with microadherence,” Clin Infect Dis, 33(9). 1567-1572. Nov.2001.
	In article
[26]	O’ Gara, J.P. and Hmphreys, H, “Staphylococcus epidermidis biofilms: Importance and implications,” J Med Microbiol, 2001, 50(7). 582-587. Jul. 2001.
	In article
[27]	Huebner, J. and Goldmann, D.A, “Coagulase-negative staphylococci: role as pathogens,” Annu Rev Med, 50. 223-236. 1999.
	In article
[28]	Maki, D.G. and Tambyah, P.A, “Engineering out the risk of infection with urinary catheters,” Emerg Infect Dis, 7(2). 1-6. March-Apr. 2001.
	In article
[29]	Pappas, P.G., Rex, J.H., Sobel, J.D., Filler, S.G., Dismukes, D.E., Walsh, T.J. and Edwards, J.E, “Guidelines for treatment of candidiasis,” Clin Infect Dis, 38(2). 161-189. Jan. 2004.
	In article
[30]	Crnich C.J. and Maki, D.G, “The promise of novel technology for the prevention of intravascular device-related bloodstream infection. I. Pathogenesis and short term devices,” Clin Infect Dis, 34(9) 1232-1242. May 2002.
	In article
[31]	El-Azizi, M., Starks, S. and Khardori, N, “Interactions of Candida albicans with other Candida species and bacteria in the biofilms,” J Apl Microbiol, 96(5). 1067-1073. 2004.
	In article
[32]	Busscher, H.J. and vand der Mei, H.C, “Use of flow chamber devices and image analysis methods to study microbial adhesion,” Methods Enzymol, 253. 455-477.1995
	In article
[33]	Lynch, R.J. and Ten Cate, J.M, “Effect of calcium glycerophosphate on demineralization in an in vitro biofilm model,” Caries Res, 40(2). 142-147. 2006.
	In article
[34]	Rosentritt, M., Hahnel, S., Groger, G., Muhlfriedel, B., Burgers, R. and Handel, G, “Adhesion of Streptococcus mutans to various dental materials in a laminar flow chamber system,” J Biomed Mater Res B Appl Biomater, 86(1). 36-44. Jul. 2008.
	In article
[35]	Xie, Q., Li, J. and Zhau, X, “Anticaries effect of compounds extracted from Galla chinensis in a multispecies biofilm model,” Oral Microbial Immunol, 23(6). 459-465. Dec. 2008.
	In article
[36]	Sjollema, J.H., Busscher, H.J. and Weer Kamp, A.H, “Deposition of oral Streptococci and polystyrene lattices onto glass in a parallel plate flow cell,” Biofouling, 1(2) 101-112. 1989
	In article
[37]	Christersson, C.E, Glantz, P.O. and Baier, R.E, “Role of temperature and shear forces on microbial detachment,” Scand. J Den Res, 96(2). 91-98. Apr. 1988.
	In article
[38]	Morisaki, H, “Measurement of the force necessary for removal of bacterial cells from aquartz plate,” Microbiol, 137. 2649-2655. 1991.
	In article